Reliable functional composition of a recombinase device family

Placeholder Show Content


This dissertation describes design principles and demonstrates applications of a recombinase device family to provide examples for how to compose reliable synthetic gene systems. I begin by introducing the functional composition challenges in building a cell cycle counter, a theoretical example of complex synthetic gene systems requiring hundreds of genetic parts. I develop novel designs for combinatorial counters that could store exponentially more states than existing examples. Using numerical simulation, I demonstrate the feasibility and limits of each design; the lack of reliably rewritable binary state devices for use in genetic counting systems is also highlighted. I then focus on experimental proof-of-concepts, failure analyses and design principles for a Recombinase Addressable Data (RAD) device built from bacteriophage integrases and excisionases. RAD devices based on Bxb1 integrase-excisionase are capable of storing state over one hundred cell generations and can be switched repeatedly without performance degradation. The tedious process of engineering a first RAD device raises the question of whether it is practical to scale such devices beyond a single bit, to implement other logical operations beyond set-reset, and to couple such devices to application-specific control signals. To address these questions, I reconfigure RAD devices to operate entirely from genomic DNA, generalize rewritable RAD principles to three other integrase-excisionase pairs, and implement single-use two input logics, buffer gates and integrase-excisionase cascades. I also demonstrate the feasibility of using competitive binding to multiplex the control of different recombination sites via the same recombinase. Finally, I show that the outcomes of composing recombinase devices with novel transcription sources can be predicted, leading to an implementation of autonomous switches driven by growth-phase dependent promoters. Taken together, the work developed here comprises an initial framework for composing complex yet reliable genetic systems using recombinase enzymes.


Type of resource text
Form electronic; electronic resource; remote
Extent 1 online resource.
Publication date 2014
Issuance monographic
Language English


Associated with Subsoontorn, Pakpoom
Associated with Stanford University, Department of Bioengineering.
Primary advisor Endy, Andrew D
Thesis advisor Endy, Andrew D
Thesis advisor Calos, Michele P
Thesis advisor Covert, Markus
Thesis advisor Horowitz, Mark (Mark Alan)
Advisor Calos, Michele P
Advisor Covert, Markus
Advisor Horowitz, Mark (Mark Alan)


Genre Theses

Bibliographic information

Statement of responsibility Pakpoom Subsoontorn.
Note Submitted to the Department of Bioengineering.
Thesis Thesis (Ph.D.)--Stanford University, 2014.
Location electronic resource

Access conditions

© 2014 by Pakpoom Subsoontorn
This work is licensed under a Creative Commons Attribution 3.0 Unported license (CC BY).

Also listed in

Loading usage metrics...